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S McFarlane

Publications and source records attributed to S McFarlane.

At least 19 recordsLinked to original sources
PubMed2006

Cortactin underpins CD44-promoted invasion and adhesion of breast cancer cells to bone marrow endothelial cells.

Using a validated tetracycline (tet)-regulated MCF7-founder (MCF7F) expression system to modulate expression of CD44 standard form (CD44s), we report the functional importance of CD44s and that of a novel transcriptional target of hyaluronan (HA)/CD44s signaling, EMS1/cortactin, in underpinning breast cancer metastasis. In functional experiments, tet-regulated induction of CD44s potentiated the migration and invasion of MCF7F cells through HA-supplemented Matrigel. EMS1/cortactin was identified by expression profiling as a novel transcriptional target of HA/CD44 signaling, an association validated by quantitative PCR and immunoblotting experiments in a range of breast cancer cell lines. The mechanistic basis underpinning CD44-promoted transcription of EMS1/cortactin was shown to be dependent upon a NFkappaB mechanism, since pharmacological inhibition of IkappaKinase-2 or suppression of p65 Rel A expression attenuated CD44-induced increases in cortactin mRNA transcript levels. Overexpression of a c-myc tagged murine cortactin construct in the weakly invasive, CD44-deficient MCF7F and T47D cells potentiated their invasion. Furthermore, the functional importance of cortactin to CD44s-promoted metastasis was demonstrated by selective suppression of cortactin in CD44-expressing MCF7F-B5 and MDA-MB-231 breast cancer cells using RNAi, which was shown to result in attenuated CD44-promoted invasion and CD44-promoted adhesion to bone marrow endothelial cells (BMECs).

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PubMed2005

Multiple signaling pathways regulate FGF-2-induced retinal ganglion cell neurite extension and growth cone guidance.

Growth cones use cues in their environment in order to grow in a directed fashion to their targets. In Xenopus laevis, fibroblast growth factors (FGFs) participate in retinal ganglion cell (RGC) axon guidance in vivo and in vitro. The main intracellular signaling cascades known to act downstream of the FGF receptor include the mitogen-activated protein kinase (MAPK), phospholipase Cgamma (PLCgamma) and phosphotidylinositol 3-kinase (PI3K) pathways. We used pharmacological inhibitors to identify the signaling cascade(s) responsible for FGF-2-stimulated RGC axon extension and chemorepulsion. The MAPK, PI3K and PLCgamma pathways were blocked by U0126, LY249002 and U73122, respectively. D609 was used to test a role for the phosphotidylcholine-PLC (PC-PLC) pathway. We determined that the MAPK and two PLC pathways are required for FGF-2 to stimulate RGC neurite extension in vitro, but the response of axons to FGF-2 applied asymmetrically to the growth cone depended only on the PLC pathways.

Animals↗
PubMed2005

Voltage-gated potassium channels regulate the response of retinal growth cones to axon extension and guidance cues.

Xenopus retinal ganglion cell growth cones express various voltage-gated potassium (Kv) channels. We showed previously that 4-aminopyridine and tetraethylammonium have different effects on the outward currents of embryonic Xenopus retinal ganglion cells. Therefore, we asked whether these Kv channel inhibitors differentially regulate the response of retinal ganglion cell growth cones to extrinsic cues. First, we tested the role of Kv channels in axon extension mediated by a substrate bound cue and found that 4-aminopyridine blocked, whereas tetraethylammonium enhanced basal extension on laminin. Yet, when the growth cones were stimulated to extend with application of soluble growth factors, both inhibitors resulted in a return to the basal extension rates observed in the presence of laminin alone. Second, we asked if Kv channels modulate the response of retinal ganglion cell growth cones to a guidance cue, the chemorepellent fibroblast growth factor-2. When presented in a gradient to one side of the growth cone, fibroblast growth factor-2 repulsed retinal ganglion cell growth cones in the presence of 4-aminopyridine but not tetraethylammonium. These data argue that tetraethylammonium- and 4-aminopyridine-sensitive Kv channels differ in the manner by which they regulate the response of retinal ganglion cell axons to extension and guidance cues. Non-ratiometric calcium imaging indicated that differences in the ability of tetraethylammonium- and 4-aminopyridine-sensitive Kv channels to regulate calcium activity within the growth cone may underlie their unique modulation of growth cone behaviour.

4-Aminopyridine↗
PubMed2005

Characterisation of the advanced glycation endproduct receptor complex in the retinal pigment epithelium.

AIMS: Advanced glycation endproducts (AGEs) accumulate with ageing and may have a significant impact on age related dysfunction of the retinal pigment epithelium (RPE). Many of the cellular effects of AGEs in other cell types are mediated through AGE binding proteins. The aim of this study was to characterise the AGE receptor complex in RPE cells in vitro and to focus on the role of the R3 component (galectin-3) as the primary effector of the complex. METHODS: Primary cultures of bovine RPE cells and the human D407 RPE cell line were exposed to AGE modified albumin. Receptor expression was determined using mRNA analysis by quantitative real time RT-PCR and protein characterisation by western blotting. Immunocytochemical analysis examined the cellular localisation of the various components of the AGE receptor complex. The role of the galectin-3 receptor component was examined by transfection and overexpression using the D407 cell line and analysis of soluble AGE-R3 by ELISA. RESULTS: All three components of the AGE receptor complex were expressed by bovine and human RPE cells. AGE exposure upregulated two components of the receptor complex and also induced significant RPE expression of VEGF mRNA (p<0.05). RPE D407 cells stably transfected to overexpress galectin-3 showed less VEGF induction. In non-transfected RPE which were exposed to AGEs, there was less soluble galectin-3 protein released into the medium (p<0.05), a response that was not evident in transfected cells. CONCLUSION: A conserved AGE receptor complex is evident in primary cultures of bovine RPE cells and also in a human cell line. These cells show a pathological response to AGE exposure, an effect which appears to be modulated by the galectin-3 component of the receptor complex.

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PubMed2004

Chloroquine causes lysosomal dysfunction in neural retina and RPE: implications for retinopathy.

Chronic use of chloroquine has been shown to induce numerous pathophysiological defects in the retina. This drug has the ability to alter pH of intracellular compartments and lysosomal function of the retinal pigment epithelium (RPE) and retinal neurons may constitute the basis of chloroquine retinopathy. The aim of the current study was to investigate pathogenic alterations in retinal cells continuously exposed to chloroquine using appropriate in vivo and in vitro models. Male hooded Lister rats were implanted with osmotic mini pumps which released chloroquine continuously over a period of seven days. The eyes were processed for electron microscopy and ultrastructural abnormalities determined in the neural retina and quantified using stereology in the retinal pigment epithelium (RPE). RPE were also exposed to chloroquine in vitro and lysosomal pH changes were investigated using a pH sensitive probe. Degradative capacity was also analysed using FITC labeled rod outer segments (ROS). Chloroquine-treated animals displayed several ultrastructural abnormalities including numerous membranous cytoplasmic bodies (MCBs) in retinal neurons. Cone photoreceptors displayed numerous MCBs although rods did not. The RPE of the treated groups all showed significantly higher numbers of lysosomal associated organelles (LAO) than the control group (p < 0.001). The in vitro experiments demonstrated chloroquine-mediated rises in lysosomal pH and an increase in lysosome/phagosome accumulation of ROS in the chloroquine treated group (p < 0.01). The current study demonstrates that chloroquine disrupts lysosomal function in retinal neurons and RPE. The evidence presented provides a clear pathogenic basis for the functional defects experienced by patients with chloroquine retinopathy.

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PubMed2003

Fibroblast growth factors redirect retinal axons in vitro and in vivo.

Growth factors have been shown previously to participate in the process of axon target recognition. We showed that fibroblast growth factor receptor (FGFR) signaling is required for Xenopus laevis retinal ganglion cell (RGC) axons to recognize their major midbrain target, the optic tectum [neuron 17 (1996), 245]. Therefore, we have hypothesized that a change in expression of a fibroblast growth factor (FGF) at the entrance of the optic tectum, the border between the diencephalon and mesencephalon, may serve as a signal to RGC axons that they have reached their target. To determine whether RGC axons can sense changes in FGF levels, we asked whether they altered their behavior upon encountering an ectopic source of FGF. We found that in vivo RGC growth cones avoided FGF-misexpressing cells along their path, and that FGF-2 directly repelled RGC growth cones in an in vitro growth cone turning assay. These data support the idea that RGC axons can sense changes in FGF levels, and as such provide a mechanism by which FGFR signaling is involved in RGC axon target recognition.

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PubMed2000

Overexpression of FGF-2 alters cell fate specification in the developing retina of Xenopus laevis.

The developing vertebrate retina produces appropriate ratios of seven phenotypically and functionally distinct cell types. Retinal progenitors remain multipotent up until the last cell division, favoring the idea that extrinsic cues direct cell fate. We demonstrated previously that fibroblast growth factor (FGF) receptors are necessary for transduction of signals in the developing Xenopus retina that bias cell fate decisions (S. McFarlane et al., 1998, Development 125, 3967-3975). However, the precise identity of the signal remains unknown. To test whether an FGF signal is sufficient to influence cell fate choices in the developing retina, FGF-2 was overexpressed in Xenopus retinal precursors by injecting, at the embryonic 16-cell stage, a cDNA plasmid encoding FGF-2 into cells fated to form the retina. We found that FGF-2 overexpression in retinal precursors altered the relative numbers of transgene-expressing retinal ganglion cells (RGC) and Müller glia; RGCs were increased by 35% and Müller glia decreased by 50%. In contrast, the proportion of retinal precursors that became photoreceptors was unchanged. Within the photoreceptor population, however, we found a twofold increase in rod photoreceptors at the expense of cone photoreceptors. These data are consistent with an endogenous FGF signal influencing cell fate decisions in the developing vertebrate retina.

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PubMed2000

A role for voltage-gated potassium channels in the outgrowth of retinal axons in the developing visual system.

Neural activity is important for establishing proper connectivity in the developing visual system. Tetrodotoxin blockade of sodium (Na(+))-dependent action potentials impairs the refining of synaptic connections made by developing retinal ganglion cells (RGCs), but does not affect their ability to get out to their target. Although this may suggest neural activity is not required for the directed extension of RGC axons, in many species developing RGCs express additional, Na(+)-independent ionic mechanisms. To test whether the ability of RGC axons to extend in a directed fashion is influenced by membrane excitability, we blocked the principal modulators of the neural activity of a neuron, voltage-dependent potassium (Kv) channels. First, we showed that RGCs and their growth cones express Kv channels when they are growing through the brain on the way to their main midbrain target, the optic tectum. Second, a Kv channel blocker, 4-aminopyridine (4-AP), was applied to the developing Xenopus optic projection. Blocking Kv channels inhibited RGC axon extension and caused aberrant routing of many RGC fibers. With the higher doses, <25% of embryos had a normal optic projection. These data suggest that Kv channel activity regulates the guidance of growing axons in the vertebrate brain.

4-Aminopyridine↗
PubMed2000

Generalized seizure in a 30-year-old man with presumed intracranial hemorrhage: a case report.

A 30-year-old man presented to the emergency department of another institution with recurrent episodes of generalized tonic-clonic seizures. He was found to be hypocalcemic and was treated with an intravenous infusion of calcium followed by Dilantin. A computed tomography scan of the head was obtained, and the patient was transferred to our institution for neurosurgical evaluation and possible intervention of what was thought to be bilateral intracranial hemorrhages. After further evaluation at our institution, the diagnoses of hypoparathyroidism associated with hypocalcemic seizures and basal ganglia calcifications were established on both clinical and biochemical grounds. This case report discusses the clinical presentation, pathogenesis, diagnostic work-up, and management of hypoparathyroidism and associated seizures, highlighting the possible diagnostic and therapeutic pitfalls that are most pertinent to the emergency physician.

Adult↗
PubMed2000

Attraction vs. repulsion: the growth cone decides.

Axons are guided through their environment in response to signals provided by extracellular cues. These cues are transduced into motile responses by the tip of the growing axon, the growth cone, and can be either repulsive or attractive in nature. Recent studies have suggested that how an axon responds to any given signal depends on the internal state of the growth cone. This review discusses these studies and their importance for understanding how nerve connections are made in the developing embryo.

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PubMed1998

Fibroblast growth factor receptor signaling in Xenopus retinal axon extension.

Fibroblast growth factor receptors (FGFRs) and N-cadherin both regulate axon extension in developing Xenopus retinal ganglion cells (RGCs). Cultured cerebellar neurons have been shown to require FGFR activity for N-cadherin-stimulated neurite outgrowth, raising the possibility that N-cadherin is a FGFR ligand. To investigate this possibility in the developing visual system, retinal neurons were transfected with a dominant-negative FGFR (XFD) and plated on purified N-cadherin substrates. XFD-expressing neurons extended markedly shorter processes than control GFP-expressing neurons, implicating a role for FGFRs in N-cadherin-stimulated neurite outgrowth. To examine whether N-cadherin and FGFRs share the same pathway or use distinct second messenger pathways, specific inhibitors of implicated signaling molecules were added to neurons stimulated by N-cadherin, basic fibroblast growth factor (bFGF), or brain-derived nerve factor (BDNF) (which stimulates RGC outgrowth by a FGFR-independent mechanism). Diacylglycerol (DAG) lipase and Ca2+/calmodulin kinase II inhibitors both significantly reduced outgrowth stimulated by N-cadherin or bFGF but not by BDNF. Furthermore, we show that inhibiting DAG lipase activity in RGC axons extending in vivo toward the optic tectum reversibly slows axon extension without collapsing their growth cones. Thus, a common second-messenger signaling pathway mediating both N-cadherin- and bFGF-stimulated neurite extension is consistent with a model in which N-cadherin directly modulates the FGFR or a model whereby both FGFR and N-cadherin regulate the same second-messenger system.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1998

A role for the fibroblast growth factor receptor in cell fate decisions in the developing vertebrate retina.

The mature vertebrate retina contains seven major cell types that develop from an apparently homogenous population of precursor cells. Clonal analyses have suggested that environmental influences play a major role in specifying retinal cell identity. Fibroblast growth factor-2 is present in the developing retina and regulates the survival, proliferation and differentiation of developing retinal cells in culture. Here we have tested whether fibroblast growth factor receptor signaling biases retinal cell fate decisions in vivo. Fibroblast growth factor receptors were inhibited in retinal precursors in Xenopus embryos by expressing a dominant negative form of the receptor, XFD. Dorsal animal blastomeres that give rise to the retina were injected with cDNA expression constructs for XFD and a control non-functional mutant receptor, D48, and the cell fates of transgene-expressing cells in the mature retina determined. Fibroblast growth factor receptor blockade results in almost a 50% loss of photoreceptors and amacrine cells, and a concurrent 3.5-fold increase in Müller glia, suggesting a shift towards a Müller cell fate in the absence of a fibroblast growth factor receptor signal. Inhibition of non-fibroblast-growth-factor-mediated receptor signaling with a third mutant receptor, HAVO, alters cell fate in an opposite manner. These results suggest that it is the balance of fibroblast growth factor and non-fibroblast growth factor ligand signals that influences retinal cell genesis.

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PubMed1997

Essential role of heparan sulfates in axon navigation and targeting in the developing visual system.

Heparan sulfate (HS) is abundant in the developing brain and is a required co-factor for many types of fibroblast growth factor (FGF) signaling in vitro. We report that some HSs, when added exogenously to the developing Xenopus optic pathway, severely disrupt target recognition causing axons from the retina to bypass their primary target, the optic tectum. Significantly, HS sidechains from a neuroepithelial perlecan variant that preferentially bind FGF-2, HS(FGF-2), cause aberrant targeting, whereas those that preferentially bind FGF-1 do not. Charge-matched fragments of HS(FGF-2) show that the mistargeting activity associates with the FGF-binding fragments. Heparitinase removal of native HSs at the beginning of optic tract formation retards retinal axon elongation; addition of FGF-2 restores axon extension but axons lose directionality. Late HS removal, after axons have extended through the tract, elicits a tectal bypass phenotype indicating a growth promoting and guidance function for native HSs. Our results demonstrate that different HS sidechains from the same core protein differentially affect axon growth in vivo, possibly due to their distinct FGF-binding preferences, and suggest that growth factors and HSs are important partners in regulating axon growth and guidance in the developing visual system.

Animals↗
PubMed1996

Inhibition of FGF receptor activity in retinal ganglion cell axons causes errors in target recognition.

Native fibroblast growth factor receptor (FGFR) function was inhibited in developing Xenopus retinal ganglion cells (RGCs) by in vivo transfection of a dominant negative FGFR. Axons expressing the dominant negative protein advanced at 60% of the normal speed, but nevertheless navigated appropriately in the embryonic optic pathway. When they neared the optic tectum, however, many axons made erroneous turns, causing them to bypass rather than enter their target. By contrast, RGC axons expressing nonfunctional FGFR mutants entered the tectum correctly. These findings demonstrate a role for FGFR signaling in the extension and targeting of RGC axons and suggest that receptor tyrosine kinase/growth factor interactions play a critical function in establishing initial connectivity in the vertebrate visual system.

Animals↗
PubMed1996

Novel stimuli for obtaining nasalance measures from young children.

The use of novel stimuli for obtaining nasalance measures in young children was the focus of this study. The subjects were 20 children without a history of communication disorders and 20 children at risk for velopharyngeal insufficiency (VPI). Each subject recited three passages; the standard Zoo Passage, and two novel stimuli that were named the Turtle Passage and the Mouse Passage. Like the Zoo Passage, the Turtle Passage contained no normally nasal consonants. The Mouse Passage was about 11% nasal consonants, which is similar to the Rainbow Passage. Statistical analysis showed no significant difference between the mean nasalance for the Zoo Passage and the Turtle Passage for either the subjects without risk of VPI (15.4% vs 15.7%) or for those at risk (30.4% vs 28.8%). Nasalance measures for the Mouse Passage were significantly higher than for either the Zoo Passage or the Turtle Passage. Listeners rated the stimuli on a 5-point equal-appearing intervals scale. The correlation coefficient between listener judgments of hypernasality and nasalance was significant for the Zoo Passage (r = 0.70) and for the Turtle Passage (r = 0.51) but not significant for the Mouse Passage (r = 0.32). Using cut-off scores of 22% for nasalance and 2.25 for hypernasality, the sensitivity for the Zoo Passage was 0.72, and for the Turtle Passage, 0.83.

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PubMed1995

FGF signaling and target recognition in the developing Xenopus visual system.

We report that the growth cones of Xenopus retinal ganglion cells express fibroblast growth factor receptors (FGFRs) and that bFGF stimulates neurite extension from cultured retinal neurons. Furthermore, bFGF is abundant in the developing optic tract but is reduced in the optic tectum. To test whether FGF signaling plays a role in axonal guidance in vivo, bFGF was exogenously applied to the developing optic pathway in "exposed brain" preparations. FGF-treated retinal axons navigate normally through the optic tract, but the majority veer aberrantly at the tectal border and bypass the target. Our results implicate FGF signaling in target recognition and suggest that diminished levels of bFGF in the tectum cause arriving axons to slow their growth.

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